Selenium-containing compounds

ABSTRACT

A novel selenium-containing compound, a method of producing the selenium-containing compound, an analysis method using the selenium-containing compound, and use of the selenium-containing compound as an antioxidant, are disclosed. A novel selenium-containing compound shown by any of chemical formulas 1 to 4 is obtained by extracting a selenium-containing compound from a sample using an organic solvent or water, and purifying the selenium-containing compound. The novel selenium-containing compound may be used for a novel analysis method, or may be used as an antioxidant.

BACKGROUND OF THE INVENTION

The present invention relates to a novel selenium-containing compound.The invention also relates to a method of producing theselenium-containing compound, use of the selenium-containing compound asan antioxidant, and an analysis method that uses the selenium-containingcompound as a standard substance.

Selenium is an essential trace element for humans. Selenium formsenzymes and proteins in vivo, and plays an important role in antioxidantreactions (Non-patent Documents 1 and 2). Selenium is abundant in algae,fish, shellfish, meat, and egg yolk (Non-patent Document 3). Selenium isalso abundant in the meat of various fish species generally consumed bythe Japanese (Non-patent Documents 1 to 4).

It is known that the dark muscle of tuna and whale meat contain seleniumin high concentration (Non-patent Documents 5 to 7). The seleniumcontent in each internal organ of Thunnus orientalis (Pacific bluefintuna) has been reported (e.g., blood (15.2 ppm), kidney (8.3 ppm),spleen (7.6 ppm), superficial dark muscle (6.1 ppm), true dark muscle(5.9 ppm), heart superficial dark muscle (4.4 ppm), liver superficialdark muscle (4.1 ppm), gills (2.6 ppm), brain (1.4 ppm), and ordinarymuscle (0.57 ppm)). Since the selenium content exceeds 4 ppm in someinternal organs, these internal organs may be used as an organicselenium source (Non-patent Document 8). However, since the biochemicalproperties of selenium in each internal organ are unclear, and it isdifficult to obtain a high-purity extract, these internal organs havenot been effectively utilized as an organic selenium source.

Since selenium is normally present in food as a selenocysteine residue(i.e., a constituent amino acid of a protein), it is considered thatselenium is digested and absorbed when absorption of the protein occurs.Therefore, the absorption of selenium in food from a digestive tract isestimated to be 50% or more (Non-patent Document 1). About 250 μg/kg ofselenium is present in the human body, and in vivo selenium homeostasisis maintained through excretion into urine (Non-patent Document 1).

The selenocysteine residue of an enzyme/protein is positioned at aselenol group (i.e., active center) of a selenoprotein. Selenoproteins(e.g., glutathione peroxidase and thioredoxin reductase that decomposeand remove active oxygen and hydroperoxides, 5′-iodothyronine deiodinasethat is involved in production of thyroid hormones, and selenoprotein Pthat is present in plasma) play an important role in an in vivoantioxidant effect (Non-patent Document 1). It has been considered basedon the above findings that organic selenium included in fish meat at ahigh concentration is a selenoprotein (e.g., glutathione peroxidase) ora peptide/amino acid thereof (Patent Document 1).

When selenium deficiency has occurred, cellular disorder occurs due toperoxides (Non-patent Document 1). The onset of cardiomyopathy (Keshandisease) (i.e., selenium deficiency disease) observed in theselenium-deficient northeast part of China is prevented byadministration of selenious acid (Non-patent Document 1). Kaschin-Beckdisease (Beck's disease) that manifests in adolescence and is observedin selenium-deficient northern China and Siberia is also caused byselenium deficiency (Non-patent Document 1).

It has been reported that selenium deficiency has a correlation withcoronary artery diseases (i.e., angina pectoris and heart infarction)based on an epidemiological study that compares the incidence of heartdiseases with the selenium level in blood. It has been reported that therate of deaths from cardiovascular diseases due to selenium deficiencyis high in eastern Finland, and a group with a serum selenium level of45 μg/1 or less has a high incidence of heart diseases (Non-patentDocument 1).

It is known that muscle pains, skin dryness, liver necrosis, and thelike are caused by selenium deficiency (Non-patent Document 1). It hasbeen reported that selenium deficiency increases the risk of cancer suchas lung cancer, large bowel cancer, prostate cancer, rectal cancer,breast cancer, and leukocythemia (Non-patent Documents 1 and 10). It hasbeen reported that the cellular immune response to cancer cells isenhanced by administering 200 μg/day of sodium selenite during surgicaltherapy or radiotherapy of cancer (Non-patent Document 8). It has alsobeen reported that selenium acts on the cancer cell signaling system tosuppress cell growth, and induces apoptosis (Non-patent Document 1).Non-patent Document 12 points out that 100 to 200 μg/day of seleniumsuppresses DNA mutation and oxidative damage due to carcinogens tosuppress the progress of cancer, but an intake of more than 400 μg/dayof selenium may be harmful. It has thus been considered that selenium iseffective for suppressing or treating cancer, or preventing recurrenceof cancer.

On the other hand, excessive intake of selenium is toxic, and may causenail deformation, unhairing, gastrointestinal injury, neuropathy, heartinfarction, acute respiratory distress, renal insufficiency, and thelike (Non-patent Document 1).

As the dietary reference intakes of selenium, the estimated averagerequirement is set to 25 (20) μg, the recommended dietary allowance isset to 30 (25) μg, and the upper limit is set to 450 (350) μg (thevalues are for adult men (the value in parentheses is the estimatedaverage requirement for adult women)) (Ministry of Health, Labour andWelfare, Nov. 22, 2004, Non-patent Document 13). Note that the estimatedaverage requirement and the recommended dietary allowance for a 30 to49-year-old male is 30 μg and 35 μg, respectively (Non-patent Document13). In Japan, the selenium upper limit is set to 100 to 450 μg Se/daytaking account of the value (800 μg Se/day) obtained by the study onEnshi, Hubei Province in China using unhairing and nail brittleness/lossas indices (Non-patent Documents 13 and 16).

In recent years, an organic selenium-containing supplement that includesinorganic selenium and selenomethionine as active ingredients has beenused to treat or prevent diseases due to selenium deficiency.Selenium-containing yeast that is obtained by culturing yeast in aculture medium including inorganic selenium, and includesselenomethionine at a high concentration has been used as an organicselenium-containing compound supply source (Non-patent Document 1). Suchselenium-containing yeast has been used to increase the selenium contentin food, a cosmetic preparation, and feed.

Organic selenium can be extracted from a raw material (e.g., meat orinternal organs of fish) having a selenium content of more than 0.5 ppmin order to supply selenium that can be efficiently used for humans,livestock, fish, and shellfish. A selenium-containing material having aselenium content of more than 5 ppm can be provided by freeze-dryingthese tissues or concentrating an extract thereof. A protein concentrateand a hydrochloric acid hydrolyzate having a total selenium content ofabout 18 to 103.5 ppm has been obtained from dark muscle (PatentDocument 1). However, since the muscles and the internal organs of largefish and carnivorous fish such as tuna contain methylmercury at arelatively high concentration of more than 0.5 ppm, an unpurified drypowder, freeze-dried product, concentrate, protease hydrolyzate, and thelike thereof contain selenium at a relatively high concentration, butalso contain methylmercury at a high concentration. Moreover, theinternal organs of fish and shellfish have a cadmium content of 1 ppm ormore. Specifically, since the dark muscles and the internal organs offish and shellfish may contain methylmercury and cadmium (toxic heavymetals), the dark muscles and the internal organs of fish and shellfishcannot be suitably used for drugs, food, feed, and the like. Therefore,it is necessary to remove toxic heavy metals by purification from anorganic selenium-containing compound derived from a fish/shellfishtissue concentrate/extract.

Non-patent Document 14 discloses a method that analyzes selenium in aliving body and food by performing thermal wet-digestion using a mixtureof nitric acid and perchloric acid, reacting the resulting product with2,3-diaminonaphthalene (DAN), and utilizing the fluorescence of4,5-benzopiaselenol (Se-DAN) produced by a complex-forming reaction withSe(IV). Non-patent Document 15 discloses a method that identifies themolecular species of inorganic and organic selenium in the environmentusing an ICP-MS that is connected to an HPLC online. However, a methodthat analyzes organic selenium-containing compounds including proteinsand amino acids in a living body and food at the same time has not beenproposed.

Selenium has been industrially used as a photoreceptor/semiconductormaterial, a red to orange pigment for glass, ceramics, and plastics, adecolorizer and an anti-foaming agent used for glass production, ametallurgical additive, and the like (Non-patent Document 16).

RELATED-ART DOCUMENT Patent Document

-   Patent Document 1: JP-A-2001-231498

Non-Patent Document

-   Non-patent Document 1: Seiichiro Himeno: Selenium, “Nutrition of    minerals/trace elements”, edited by Tsugumi Suzuki and Osamu Wada,    DAI-ICHI SHUPPAN Co., Ltd., pp. 423-445, 1994-   Non-patent Document 2: Gerald F., Jr. Combs: Selenium in global food    systems, British Journal of Nutrition, 85(5), pp. 517-547 (2001)-   Non-patent Document 3: Gerald F., Jr. Combs: The Role of Selenium in    Nutrition, Academic Press, pp. 1-532, 1986-   Non-patent Document 4: Yasuo Suzuki (editor), Table of trace element    contents in Japanese foodstuffs, DAI-ICHI SHUPPAN Co., Ltd., pp.    1-169, 1993-   Non-patent Document 5: Satoshi Arima and Katsuo Nagakura, Mercury    and selenium content in toothed whales, Nippon Suisan Gakkaishi, 45,    pp. 623-626, 1979-   Non-patent Document 6: Yumiko Yamashita, Food chemical research on    essential trace elements in fish, Marine product processing research    result/program summary (1993), National Research Institute of    Fisheries Science, Fisheries Agency, pp. 10-11, February 1994-   Non-patent Document 7: Yumiko Yamashita, Food chemical research on    essential trace elements in fish, Marine product processing research    result/program summary (1996), National Research Institute of    Fisheries Science, Fisheries Agency, pp. 28-29, February 1997-   Non-patent Document 8: L. Kiremidjian-Schumacher and M. Roy, Effect    of selenium on the immunocompetence of patients with head and neck    cancer and on adoptive immunotherapy of early and established    lesions, Biofactors, 14 (1-4), pp. 161-168 (2001)-   Non-patent Document 9: J. T. Salonen, G Alfthan, J. K. Huttunen, J.    Pikkarainen, and P. Puska, Association between cardiovascular death    and myocardial infarction and serum selenium in a watched-pair    longitudinal study, Lancet, 2, pp. 175-179 (1982)-   Non-patent Document 10: Yoshikazu Kawakami, Vitamin E and    respiratory diseases, CLINICIAN, 356, pp. 63-66 (1986)-   Non-patent Document 11: A. Ghose, J. Fleming, and P R. Harrison,    Selenium and signal transduction: roads to cell death and    anti-tumour activity, Biofactors, 14 (1-4), pp. 127-133 (2001)-   Non-patent Document 12: K. El-Bayoumy, The protective role of    selenium on genetic damage and on cancer., Mutat. Res. 475 (1-2),    pp. 123-139 (2001)-   Non-patent Document 13: Ministry of Health, Labour and Welfare,    Dietary Reference Intakes for Japanese, Nov. 22, 2004-   Non-patent Document 14: J. H. Watkinson, Fluorometric determination    of selenium in biological material with 2,3-diaminonaphthalene.    Anal. Chem., 38(1), pp. 92-97 (1966)-   Non-patent Document 15: H. Ge, X. J. Cai, J. F. Tyson, P. C.    Uden, E. R. Denoyer, and E. Block, Anal. Commun. 33 (1996) 279-   Non-patent Document 16: Selenium and its compounds, Chemical    Management Center, National Institute of Technology and Evaluation,    http://www.safe.nite.go.jp/management/search/Fundamental/76

SUMMARY OF THE INVENTION

An object of the invention is to provide a selenium-containing compound.Another object of the invention is to provide a method of producing theselenium-containing compound, use of the selenium-containing compound asan antioxidant, and an analysis method that uses the selenium-containingcompound as a standard substance.

The inventors of the invention conducted extensive studies in order toachieve the above objects, and found a novel selenium-containingcompound having a strong in vivo antioxidant effect. The novelselenium-containing compound was obtained by extracting a seleniumconcentrate from a biological sample (e.g., fish) using an organicsolvent, and isolating/purifying a selenium-containing compound in theselenium concentrate by chromatography.

The inventors also found that the selenium-containing compound can beisolated and quantitatively determined by column chromatography, massspectrometry, or the like, and made it possible to provide an analysismethod that uses the selenium-containing compound as a standardsubstance. A protein to which selenium is specifically bound, or aselenium-containing compound distributed in a living body and food, canbe analyzed by this analysis method.

Specifically, the invention relates to the following selenium-containingcompound and the like recited in (1) to (58).

(1) A selenium-containing compound shown by the following chemicalformula 1,

wherein R is absent, or represents hydrogen or an organic compound.(2) The selenium-containing compound according to (1), theselenium-containing compound being shown by the following chemicalformula 2, 3, or 4.

(3) A method of analyzing a selenium-containing compound includinganalyzing a selenium-containing compound using the selenium-containingcompound according to (1) or (2) as a standard substance.(4) A drug, a functional food, a nutritional supplement, a foodadditive, an animal drug, a feed additive, or a cosmetic preparationincluding the selenium-containing compound according to (1) or (2).(5) A medium additive including the selenium-containing compoundaccording to (1) or (2).(6) An antioxidant including the selenium-containing compound accordingto (1) or (2).(7) A methemoprotein formation inhibitor including theselenium-containing compound according to (1) or (2).(8) An artificial blood that includes hemoglobin or an oxygen carrierhaving an effect equivalent to that of hemoglobin, a transfusionpreparation, or a tissue preservation solution produced using theselenium-containing compound according to (1) or (2).(9) A fluorescent substance, a UV absorber, or a chemical modifierincluding the selenium-containing compound according to (1) or (2).(10) A selenium concentrate including the selenium-containing compoundaccording to (1) or (2).(11) A method of producing the selenium-containing compound according to(1) or (2), the method including extracting the selenium-containingcompound according to (1) or (2) from a sample using an organic solventor water.(12) The method according to (11), including extracting theselenium-containing compound according to (1) or (2) from the sampleusing a hydrophilic organic solvent, followed by extraction with asolvent including a hydrophobic organic solvent.(13) The method according to (11), wherein the hydrophilic organicsolvent is at least one compound selected from ethanol, methanol,acetone, and acetonitrile, and the hydrophobic organic solvent is atleast one compound selected from diethyl ether, tetrahydrofuran,cyclohexane, and dichloromethane.(14) The method according to (12), wherein the hydrophilic organicsolvent is at least one compound selected from ethanol, methanol,acetone, and acetonitrile, and the hydrophobic organic solvent is atleast one compound selected from diethyl ether, tetrahydrofuran,cyclohexane, and dichloromethane.(15) The method according to (11), further including reducing the samplethat includes the selenium-containing compound according to (1) or (2),or a selenium concentrate that includes the selenium-containing compoundaccording to (1) or (2), using a reducing agent.(16) The method according to (12), further including reducing a samplethat includes the selenium-containing compound according to (1) or (2),or a selenium concentrate that includes the selenium-containing compoundaccording to (1) or (2), using a reducing agent.(17) The method according to (13), further including reducing a samplethat includes the selenium-containing compound according to (1) or (2),or a selenium concentrate that includes the selenium-containing compoundaccording to (1) or (2), using a reducing agent.(18) The method according to (14), further including reducing a samplethat includes the selenium-containing compound according to (1) or (2),or a selenium concentrate that includes the selenium-containing compoundaccording to (1) or (2), using a reducing agent.(19) The method according to (16), wherein the reducing agent is a thiolreducing agent.(20) The method according to (17), wherein the reducing agent is a thiolreducing agent.(21) The method according to (18), wherein the reducing agent is a thiolreducing agent.(22) The method according to (19), wherein the reducing agent is a thiolreducing agent.(23) The method according to (11), further including concentrating aselenium concentrate using an ion-exchange resin.(24) The method according to (12), further including concentrating aselenium concentrate using an ion-exchange resin.(25) The method according to (13), further including concentrating aselenium concentrate using an ion-exchange resin.(26) The method according to (14), further including concentrating aselenium concentrate using an ion-exchange resin.(27) The method according to (15), further including concentrating aselenium concentrate using an ion-exchange resin.(28) The method according to (16), further including concentrating aselenium concentrate using an ion-exchange resin.(29) The method according to (17), further including concentrating aselenium concentrate using an ion-exchange resin.(30) The method according to (18), further including concentrating aselenium concentrate using an ion-exchange resin.(31) The method according to (19), further including concentrating aselenium concentrate using an ion-exchange resin.(32) The method according to (20), further including concentrating aselenium concentrate using an ion-exchange resin.(33) The method according to (21), further including concentrating aselenium concentrate using an ion-exchange resin.(34) The method according to (22), further including concentrating aselenium concentrate using an ion-exchange resin.(35) The method according to (11), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(36) The method according to (12), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(37) The method according to (13), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(38) The method according to (14), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(39) The method according to (15), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(40) The method according to (16), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(41) The method according to (17), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(42) The method according to (18), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(43) The method according to (19), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(44) The method according to (20), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(45) The method according to (21), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(46) The method according to (22), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(47) The method according to (23), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(48) The method according to (24), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(49) The method according to (25), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(50) The method according to (26), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(51) The method according to (27), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(52) The method according to (28), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(53) The method according to (29), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(54) The method according to (30), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(55) The method according to (31), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(56) The method according to (32), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(57) The method according to (33), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).(58) The method according to (34), further including purifying aselenium concentrate by high performance liquid chromatography (HPLC).

The novel selenium-containing compound obtained by the invention may beused as a standard substance for analyzing biological components andfood. The selenium content in a living body and food can be measured foreach chemical form by analyzing a selenium-containing protein, aselenium-containing peptide, a selenium-containing amino acid, andinorganic selenious acid in a living body and food using theselenium-containing compound. Since the selenium-containing compoundaccording to the invention is safe and has high bioavailability ascompared with selenious acid and the like, selenium can be efficientlyincorporated in a living body such as humans, livestock, fish, andshellfish. Since the selenium-containing compound according to theinvention exhibits an in vivo antioxidant effect equal to or higher thanthat of known antioxidants, the selenium-containing compound may be usedas an active ingredient of a drug, a functional food, a nutritionalsupplement, a food additive, an animal drug, a feed additive, a cosmeticpreparation, and the like, as an antioxidant that improves an in vivoantioxidant effect in a redox pathway that involves selenium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing a process of purifying a selenium-containingcompound by HPLC from a selenium concentrate derived from the darkmuscle of Thunnus obesus (Example 1). FIG. 1 shows a process thatisolates a selenium concentrate derived from the dark muscle of Thunnusobesus.

FIG. 2 is a view showing isolation of a selenium-containing compound bygel-filtration chromatography and the chemical structure analysisresults for the novel selenium-containing compound (Example 1).

FIG. 3 is a view showing the ¹H NMR analysis results for aselenium-containing compound (Example 4).

FIG. 4 is a view showing the electrospray ionization mass spectrometry(ESI-MS, spectrum 1) results and the electrospray ionization tandem massspectrometric analysis (ESI-MS/MS, spectrum 2) results for aselenium-containing compound oxidized dimer (Example 4).

FIG. 5 is a view showing the liquid chromatography-inductively coupledplasma mass spectrometry (HPLC-ICP-MS) results for a selenium-containingcompound (Example 5).

FIG. 6 is a view showing the HPLC-ICP-MS results for aselenium-containing compound (Example 5).

FIG. 7 is a view showing the HPLC-ICP-MS results for aselenium-containing compound (Example 5).

FIG. 8 is a view showing the in vivo antioxidant effect of aselenium-containing compound purified from the dark muscle using thecell proliferation rate (Example 5).

FIG. 9 is a view showing the cell proliferation-promoting effect of aselenium-containing compound (Example 5).

FIG. 10 is a view showing the rate of metmyoglobin formation ofhemoglobin collected from red cells (Example 5).

FIG. 11 is a view showing the active oxygen level in a hemoglobinsolution collected from red cells (Example 5).

FIG. 12 is a view showing iron and selenium detected from myoglobin(Example 5).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

A selenium-containing compound according to one embodiment of theinvention includes a skeleton shown by the chemical formula 1, wherein Rrepresents hydrogen or an organic compound. For example, theselenium-containing compound may be a selenium-containing compound shownby the chemical formula 2(3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate) or aselenium-containing compound shown by the chemical formula 3(3-(2-selenoxo-2,3-dihydro-1H-imidazol-4-yl)-2-(trimethylammonio)propanoate).The selenium-containing compound shown by the chemical formula 2 or 3 isa monomer of the selenium-containing compound. The selenium-containingcompound shown by the chemical formula 2 has a molecular structure inwhich a selenol group is bonded to a carbon atom at position 2 of apyrimidine ring. Since a selenol group forms a tautomer that is in anequilibrium state with a selenoketone group, the selenium-containingcompound may have a selenol-type chemical form (chemical formula 2) anda selenoketone-type chemical form (chemical formula 3) in a solutionstate depending on the solvent conditions. An oxidized dimer is easilyformed in a non-polar solvent in which the equilibrium shifts to theselenol isomer, and the chemical equilibrium shifts to the selenoketoneisomer in a polar solvent so that the selenium-containing compound ismainly present as a monomer.

The selenium-containing compound according to one embodiment of theinvention also includes a dimer shown by the chemical formula 4(oxidized dimer:3,3′-(2,2′-diselanediylbis(1H-imidazole-5,2-diyl))bis(2-(trimethylammonio)propanoate))in which compounds (basic units) having a molecular structure in which aselenol group and a trimethylammonium group are bonded to a pyrimidinering form a diselenide through the selenol groups.

The selenium-containing compound according to one embodiment of theinvention also includes selenium-containing compounds in whichergothioneine, glutathione, cysteine, acetylcysteine, homocysteine,methylmercury, or a thiol compound considered to be produced in vivo isbonded to R, and metal or polymer materials to which theselenium-containing compound is bonded through the selenol group, inaddition to the selenium-containing compounds shown by the chemicalformulas 2 to 4.

A method of analyzing a selenium-containing compound according to oneembodiment of the invention includes analyzing a selenium-containingprotein (e.g., glutathione peroxidase or selenoprotein P), aselenium-containing peptide, a selenium-containing amino acid (e.g.,selenocystine or selenomethionine), an organic or inorganic seleniousacid (e.g., the selenium-containing compound according to one embodimentof the invention (selenoneine)), or the like included in a living body,food, or drink (i.e., measurement target) using the selenium-containingcompound according to one embodiment of the invention as a standardsubstance.

For example, the selenium-containing compound according to oneembodiment of the invention is isolated and purified by chromatography,and introduced into an ICP-MS online to ionize selenium. Aselenoprotein, a selenium-containing amino acid, selenious acid, andorganic and inorganic selenium included in the measurement target canthus be analyzed and measured at the same time (see FIGS. 5 and 6).

A selenium-containing compound included in a living body or food may beisolated by chromatography under arbitrary conditions insofar as aselenoprotein, a selenium-containing amino acid, selenious acid, andorganic and inorganic selenium can be analyzed and measured. Forexample, the selenium content in each chemical form may bequantitatively analyzed by isolating a living tissue extract using agel-filtration column (“Ultrahydrogel 120” manufactured by Waters Co.,inner diameter: 7.8 mm, column length: 300 mm) that is equilibrated with0.1 M ammonium formate (see FIG. 7).

The selenium-containing compound according to one embodiment of theinvention may be included in a drug, a functional food, a nutritionalsupplement, a food additive, an animal drug, a feed additive, a cosmeticpreparation, a medium additive, an antioxidant, and the like as anactive ingredient. The selenium-containing compound according to oneembodiment of the invention may be included in a methemoproteinformation inhibitor, an artificial blood including hemoglobin or anoxygen carrier having an effect equivalent to that of hemoglobin, atransfusion preparation, a tissue preservation solution, a fluorescentsubstance, a UV absorber, a chemical modifier, and the like as an activeingredient. A selenium concentrate including a selenium-containingcompound obtained when producing the selenium-containing compoundaccording to one embodiment of the invention may also be used as a rawmaterial including an active ingredient.

The drug that includes the selenium-containing compound according to oneembodiment of the invention may be a preventive preparation, atherapeutic agent, and the like that is effective for diseasesconsidered to be caused by selenium deficiency as an antioxidant thatimproves an in vivo antioxidant effect in a redox pathway that involvesselenium. Examples of diseases that are considered to be caused byselenium deficiency include cancers such as lung cancer, prostatecancer, and large bowel cancer, heart diseases, diabetes, and the like.

For example, it is known that the onset of (or death from)cardiomyopathy (Keshan disease) observed in a selenium-deficient area inChina at an altitude of 1000 meters or more is improved by intake ofselenious acid. Cardiomyopathy (Keshan disease) is considered to occurwhen the selenium-containing compound according to one embodiment of theinvention bound to myoglobin and hemoglobin becomes deficient underlow-oxygen conditions so that auto-oxidation of heme iron and radicalformation occur to a large extent. It has been reported that the rate ofdeaths from cardiovascular diseases due to selenium deficiency is highin eastern Finland, and a group with a serum selenium level of 45 μg/1or less has a high incidence of heart diseases (Non-patent Document 1).

It is conjectured that the risk of heart diseases can be reduced byimproving an in vivo antioxidant effect in a redox pathway that involvesselenium via administration of the drug that includes theselenium-containing compound according to one embodiment of theinvention.

It is known that the selenium intake level and the cancer mortality ratehave a negative correlation. Studies conducted in Finland and the UnitedStates suggest that the cancer mortality rate of a group with a lowserum selenium level increases by a factor of 2 to 3 (Non-patentDocument 1). It is conjectured that the risk of cancer due to seleniumdeficiency can be reduced by improving an in vivo antioxidant effect ina redox pathway that involves selenium by providing the drug thatincludes the selenium-containing compound.

The animal drug that includes the selenium-containing compound accordingto one embodiment of the invention may be a preventive preparation, atherapeutic agent, and the like effective for animal diseases that areconsidered to be caused by selenium deficiency by improving an in vivoantioxidant effect in a redox pathway that involves selenium. Thesepreventive preparations, therapeutic agents, and the like can beprovided in the same manner as the drug that includes theselenium-containing compound according to one embodiment of theinvention.

The functional food that includes the selenium-containing compoundaccording to one embodiment of the invention includes any type of foodthat includes the selenium-containing compound according to oneembodiment of the invention, and is useful for preventing or reducing(treating) symptoms of diseases that are considered to be caused byselenium deficiency by improving an in vivo antioxidant effect in aredox pathway that involves selenium, and it is permitted to specify tothat effect.

The nutritional supplement that includes the selenium-containingcompound according to one embodiment of the invention includes any typeof nutritional supplement that includes the selenium-containing compoundaccording to one embodiment of the invention, and prevents seleniumdeficiency. Examples of the nutritional supplement include nutritionalsupplements and the like to which the selenium-containing compoundaccording to one embodiment of the invention is added in an amount ofabout 50 to about 200 μg (i.e., an amount necessary for preventingselenium deficiency).

The food additive that includes the selenium-containing compoundaccording to one embodiment of the invention, or the feed additive thatincludes the selenium-containing compound according to one embodiment ofthe invention, includes any type of food additive or feed additive thatincludes the selenium-containing compound according to one embodiment ofthe invention, and is added to food or feed in order to prevent seleniumdeficiency by improving an in vivo antioxidant effect in a redox pathwaythat involves selenium.

The cosmetic preparation that includes the selenium-containing compoundaccording to one embodiment of the invention includes any type ofcosmetic preparation that includes the selenium-containing compoundaccording to one embodiment of the invention, and is used to solvebeauty problems by improving an in vivo antioxidant effect in a redoxpathway that involves selenium.

The selenium-containing compound according to one embodiment of theinvention is a selenium source that has very low cytotoxicity ascompared with sodium selenite and selenomethionine that have been usedfor the above applications, does not include a toxic heavy metal, and iseasily absorbed into a living body. Therefore, the selenium-containingcompound according to one embodiment of the invention is very useful.

It was confirmed by a chemical test that the selenium-containingcompound according to one embodiment of the invention is a substancethat has a very strong antioxidant ability, and has a radical-scavengingability higher than that of a water-soluble vitamin E derivative“Trolox” (registered trademark)(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) by a factor ofabout 500. It is conjectured that the selenium-containing compoundaccording to one embodiment of the invention has a DNA repair effect.Therefore, the selenium-containing compound according to one embodimentof the invention may be used as an antioxidant, and may also be used toprovide a functional food, a nutritional supplement, a food additive, ora cosmetic preparation that achieves an anti-aging effect by improvingan in vivo antioxidant effect in a redox pathway that involves selenium.

Selenious acid, selenocystine, selenomethionine, and the like have alsobeen used for administration tests on living organisms. Theselenium-containing compound according to one embodiment of theinvention is a safe and useful substance that has high bioavailabilityand very low cytotoxicity as compared with selenious acid,selenocystine, selenomethionine, and the like.

The medium additive that includes the selenium-containing compoundaccording to one embodiment of the invention includes any type of mediumadditive that can be added as a selenium source or an antioxidant to aculture medium used when artificially culturing animal cells, plantcells, and microorganisms.

The selenium-containing compound according to one embodiment of theinvention is specifically introduced into cells (e.g., vesselendothelial cells or red cells), and has low cytotoxicity. Therefore,the selenium-containing compound according to one embodiment of theinvention may be used as a medium additive that is added to a serum-freemedium or a low-serum medium for animal cells instead of selenious acid.

The selenium-containing compound according to one embodiment of theinvention is an organic cationic betaine compound having a chemicalstructure similar to that of ergothioneine, carnitine, and the like, ispromptly introduced into cells from a culture medium in a cell culturetest, and promotes cell growth. It is conjectured that transport insideand outside the cells is controlled by an organic cation/carnitinetransporter (OCTN) (I. Tamai et al., Mol. Pharm. 1, pp. 57-66 (2003),and D. Grundemann et al., Discovery of the ergothioneine transporter,PNAS, 102, pp. 5256-5261 (2005)).

The methemoprotein formation inhibitor that includes theselenium-containing compound according to one embodiment of theinvention includes any type of substance that includes theselenium-containing compound according to one embodiment of theinvention, and inhibits methemoprotein formation.

The transfusion preparation, the tissue preservation solution, or theartificial blood using the selenium-containing compound according to oneembodiment of the invention includes any type of transfusion preparationor tissue preservation solution that includes the selenium-containingcompound according to one embodiment of the invention, or any type ofartificial blood that includes hemoglobin or an oxygen carrier having aneffect equivalent to that of hemoglobin. Examples of the oxygen carrierhaving an effect equivalent to that of hemoglobin include ahemoglobin-containing ribosome, a porphyrin metal complex-albumincomplex, a polyethylene glycolated (PEG) porphyrin metal complex-albumincomplex, crosslinked hemoglobin, a hemoglobin polymer, a polyethyleneglycolated hemoglobin polymer, and the like.

Since the selenium-containing compound according to one embodiment ofthe invention functions as an antioxidant or a heme iron auto-oxidationinhibitor, the selenium-containing compound according to one embodimentof the invention may be used when storing blood, organs, or artificialblood.

Nutrition therapy (e.g., total parenteral nutrition) has been used whentreating digestive system diseases. It has been reported that nutritiontherapy results in deficiency of trace elements (Non-patent Document 1).In this case, when adding the selenium-containing compound according toone embodiment of the invention to a component of the transfusionpreparation, the selenium-containing compound is circulated through thebody via blood, and introduced into tissues and cells, so that seleniumdeficiency can be prevented.

The fluorescent substance, the UV absorber, or the chemical modifierthat includes the selenium-containing compound according to oneembodiment of the invention includes any type of substance that includesthe selenium-containing compound according to one embodiment of theinvention, and can be used as a fluorescent substance, a UV absorber, ora chemical modifier. Since the selenium-containing compound according toone embodiment of the invention includes a selenol group having higherreactivity than that of a thiol group, and has fluorescent and UVabsorption characteristics, and the like, the selenium-containingcompound according to one embodiment of the invention may be used asvarious industrial materials (e.g., fluorescent substance, UV absorber,or chemical modifier).

The method of producing the selenium-containing compound according toone embodiment of the invention includes extracting theselenium-containing compound shown by any of the chemical formulas 1 to4, or a selenium concentrate including the selenium-containing compoundshown by any of the chemical formulas 1 to 4 from a sample using wateror an organic solvent.

The selenium concentrate including the selenium-containing compoundaccording to one embodiment of the invention refers to a seleniumconcentrate including the selenium-containing compound shown by any ofthe chemical formulas 1 to 4 obtained when producing theselenium-containing compound according to one embodiment of theinvention. For example, the selenium concentrate including theselenium-containing compound according to one embodiment of theinvention may be obtained by obtaining an extract from a sample using anorganic solvent or water, and concentrating the extract using a rotaryevaporator or the like. When the selenium concentrate including theselenium-containing compound according to one embodiment of theinvention is a solution, it is preferable that the selenium concentrateincludes the selenium-containing compound according to one embodiment ofthe invention in an amount of 5 μg/ml or more. The selenium concentratemay also include ergothioneine that has chemical properties similar tothose of the selenium-containing compound according to one embodiment ofthe invention.

In the method of producing the selenium-containing compound according toone embodiment of the invention, the selenium-containing compound may beextracted from the sample using water or an organic solvent by immersingthe sample in an organic solvent (e.g., acetone, ethanol, or methanol),water, or the like, optionally crushing the tissues in the solvent, andcollecting a soluble fraction including the selenium-containing compoundshown by any of the chemical formulas 1 to 4 dissolved in the solvent bycentrifugation, filtration, or the like.

The above step may further include concentrating the collected solublefraction by vacuum concentration, membrane separation, electrodialysis,chromatography, or freeze-drying.

The type of organic solvent used for extraction, the number ofextraction operations, and the like are not particularly limited insofaras the selenium-containing compound shown by any of the chemicalformulas 1 to 4 or a selenium concentrate including theselenium-containing compound shown by any of the chemical formulas 1 to4 can be obtained from the sample. It is preferable that the methodinclude extracting the selenium-containing compound from the sampleusing a hydrophilic organic solvent, followed by extraction with asolvent including a hydrophobic organic solvent. For example, the purityof the selenium-containing compound to be extracted increases byextracting the selenium-containing compound from the sample usingethanol, methanol, or acetone (i.e., hydrophilic organic solvent),followed by extraction with a mixture of acetonitrile (i.e., hydrophilicorganic solvent) and diethyl ether (i.e., hydrophobic organic solvent)(i.e., a solvent including a hydrophobic organic solvent).

As the hydrophilic organic solvent, a known hydrophilic organic solventmay be used. For example, the hydrophilic organic solvent may be atleast one compound selected from ethanol, methanol, acetone, andacetonitrile. As the hydrophobic organic solvent, a known hydrophobicorganic solvent may be used. For example, the hydrophobic organicsolvent may be at least one compound selected from diethyl ether,tetrahydrofuran or dichloromethane. The solvent including a hydrophobicorganic solvent may be an arbitrary solvent including such a hydrophobicorganic solvent, and includes a mixed solvent of a hydrophilic organicsolvent and a hydrophobic organic solvent, and a solvent that includesonly a hydrophobic organic solvent.

The method of producing the selenium-containing compound according toone embodiment of the invention preferably further includes reducing thesample or the selenium concentrate using a reducing agent since theselenium-containing compound may be strongly bonded (coordinated) toheme iron to form a heme iron complex. The heme iron of the heme ironcomplex may be removed by extraction with an organic solvent such asacetonitrile, but can be reliably removed using a reducing agent.

The sample or a selenium concentrate including the selenium-containingcompound obtained from the sample may be reduced using a reducing agent.It is preferable to reduce the sample or the selenium concentrate usinga thiol reducing agent. Examples of the thiol reducing agent includedithiothreitol, 2-mercaptoethanol, glutathione, and the like.

The selenium-containing compound can be isolated by the reduction stepusing the reducing agent, so that the extraction efficiency from thesample can be increased, and methylmercury (toxic component) can beremoved.

The method of producing the selenium-containing compound according toone embodiment of the invention preferably includes concentrating theselenium concentrate using an ion-exchange resin, or concentrating theselenium concentrate by high performance liquid chromatography (HPLC).

The concentration of the selenium-containing compound in the seleniumconcentrate can be increased by concentrating the selenium concentrateusing an ion-exchange resin. The selenium-containing compound shown byany of the chemical formulas 1 to 4 can be obtained by concentrating theselenium concentrate by HPLC.

The selenium-containing compound according to one embodiment of theinvention can be purified to high purity by isolating (separating) theselenium concentrate including the selenium containing compound obtainedby extraction using water or an organic solvent by chromatography (e.g.,silica gel column chromatography, ion-exchange column chromatography,C18 reversed-phase column chromatography, or thin-layer chromatography).The selenium-containing compound according to one embodiment of theinvention thus purified to high purity includes selenium in an amount of20 to 29% (200,000 to 290,000 ppm).

The sample from which the selenium-containing compound according to oneembodiment of the invention is extracted is not particularly limitedinsofar as the sample includes the selenium-containing compound shown byany of the chemical formulas 1 to 4. For example, theselenium-containing compound was detected from squid tissues, fishtissues, bird tissues, and mammal tissues. Therefore, these tissues maybe used as the sample optionally in the form of a dried product or apowder.

A large amount of selenium-containing compound is included in the ediblepart and fishery processing residues (e.g., dark muscle, spleen,hepatopancreas, heart, and blood) of fish and shellfish (e.g., tuna),the internal organs of salmon, Beryx splendens, Todarodes pacificus, andthe like, the internal organs of whales, livestock, and domestic fowl,and the like. Therefore, it is also preferable to use the tissues ofthese internal organs and the like as the sample optionally in the formof a dried product or a powder.

Specifically, the dark muscle of tuna such as Thunnus orientalis,Thunnus maccoyii, Thunnus obesus, and Thunnus alalunga, whale meat suchas the lean meat of Globicephala macrorhynchus, tuna blood, thehepatopancreas of tuna such as Thunnus orientalis, and the like may beused.

A product that is chemically or enzymatically synthesized from aminoacids (e.g., histidine, hercynine, and ergothioneine) or betaines thatare considered to be located in the same metabolic pathway as theselenium-containing compound according to one embodiment of theinvention may also be used as the sample.

The sample may also be microorganisms, cultured cells, animals, orplants that biosynthesize or store the selenium-containing compoundaccording to one embodiment of the invention.

The invention is further described below by way of examples. Note thatthe invention is not limited to the following examples.

EXAMPLES Example 1

Extraction of selenium-containing compound

1. Sample

1) Dark muscle: The dark muscle of Thunnus orientalis, Thunnus maccoyii,Thunnus obesu, or Thunnus alalunga was used as tuna dark muscle. Thedark muscle of Xiphias gladius was used as marlin dark muscle.

2) Whale meat: The lean meat of Globicephala macrorhynchus was used.

3) Tuna blood: The blood of Thunnus orientalis was used.

4) Tuna hepatopancreas: The hepatopancreas of Thunnus orientalis wasused.

2. Measurement of Selenium Content

The selenium content in the selenium-containing compound or the seleniumconcentrate including the selenium-containing compound was measured bythe following fluorometry.

<Fluorometry>

10 to 100 μl of a sample solution was wet-ashed in a graduated stoppertest tube at 210° C. for 2 hours together with 1 ml of a mixed acid(nitric acid:perchloric acid:sulfuric acid=1:2) (hereinafter referred toas “mixed acid”). After the addition of 0.2 ml of a saturated ammoniumoxalate aqueous solution, the mixture was heated in a water bath at 100°C. for 5 minutes.

After the addition of 0.2 ml of 6N hydrochloric acid, the mixture washeated in a water bath at 100° C. for 30 minutes. After cooling themixture with water, 0.2 ml of 0.1 M disodium ethylenediaminetetraacetatewas added. The pH of the mixture was adjusted to 1.0 to 1.5 using a 6Nsodium hydroxide aqueous solution. After the addition of 1 ml of DAN (1mg/ml) dissolved in 0.1N hydrochloric acid, the mixture was heated at50° C. for 20 minutes.

After cooling the mixture with water, the mixture was shaken togetherwith 1 ml of cyclohexane. The fluorescence of the cyclohexane layer wasthen measured (excitation light: 379 nm, fluorescence wavelength: 521nm). A calibration curve was drawn by fluorometry using a blank and 5,10, 50, and 100 μl of a 1 mg/l selenium standard solution, and theselenium content in the sample was calculated.

3. Extraction Method

1) Extraction of Selenium-Containing Compound from Tuna Dark Muscle orMarlin Dark Muscle

Step 1: Extraction of Heme Iron Complex of Selenium-Containing Compound

The dark muscle of Thunnus orientalis, the dark muscle of Thunnusmaccoyii, the dark muscle of Thunnus obesus, the dark muscle of Thunnusalalunga, or the dark muscle of Xiphias gladius was minced to athickness of 1 cm. The minced dark muscle was put into a polyethylenebottle with a lid. After the addition of a 3-fold amount of methanolcooled to 0° C. or less, the mixture was allowed to stand at 0° C. for 1week or more.

An equal amount of 100% ethanol (cooled to −40° C. or less) was added to1 l of the methanol extract from which the meat had been removed, andthe precipitate was removed by centrifugation (6000×g, 20 min). Theremaining extract was evaporated to dryness using a rotary evaporator toobtain a selenium concentrate (1).

After the addition of an adequate amount (about 100 to 500 ml) of coldmethanol to the selenium concentrate (1), the mixture was sufficientlystirred, and filtered (filter paper No. 3 manufactured by Toyo RoshiCo., Ltd.; hereinafter the same). The amount of cold methanol may beappropriately determined since the mixture is evaporated afterextraction. In this example, cold methanol was added so that asuspension state that allows filtration and extraction was achievedwhile visually observing that a pigment was extracted.

After the addition of an equal amount of cold ethanol to the extract,the mixture was evaporated using a rotary evaporator to obtain aselenium concentrate (2). After the addition of 10 to 100 ml of coldethanol to the selenium concentrate (2), the mixture was sufficientlyshaken. The precipitate was then removed by filtration or centrifugation(6000×g, 20 min). The extract was collected into a recovery flask, andevaporated using a rotary evaporator to obtain a selenium concentrate(3). The selenium concentrate (3) was dissolved in about 10 ml of coldwater, and the precipitate was removed by centrifugation (10,000×g, 5min).

The color of the selenium concentrate (3) thus obtained was orange, red,or brown. The selenium concentrate (3) was collected as a heme ironcomplex of the selenium-containing compound. The color of the heme ironcomplex of the selenium-containing compound differed depending on thetype of the sample, the fish species, the pigment content, the storagestate (degree of oxidation) of the sample, the extraction temperature,the effect of the reducing agent, and the like.

The selenium content in a dried product obtained by evaporating theselenium concentrate (3) to dryness using a centrifugal evaporator was16 to 152 mg/kg (dark muscle of Thunnus orientalis: 16 mg, dark muscleof Thunnus maccoyii: 17 mg, dark muscle of Thunnus obesus: 152 mg, darkmuscle of Thunnus alalunga: 63 mg, and dark muscle of Xiphias gladius:21 mg). The main selenium-containing compounds included in the seleniumconcentrate (3) were selenoketone-type selenium-containing compounds(chemical formula 3) in which heme iron was bonded to selenium.

Step 2: Removal of Heme Iron

The heme iron complex of the selenium-containing compound included inthe selenium concentrate (3) obtained by the step 1 was reduced using areducing agent to remove heme iron. The yield of the selenium-containingcompound was thus increased.

Specifically, dithiothreitol (final concentration: 0.1%) was added tothe selenium concentrate (3) derived from the dark muscle of Thunnusobesus obtained by the step 1 to obtain a reaction solution. A 5-foldamount of acetonitrile-diethyl ether mixture (volume ratio: 5:1) wasadded to the reaction solution, and an organic layer including theselenium-containing compound was collected via a two-phase separation.The extraction step and the concentration step were repeated severaltimes until the extract became transparent and clear to remove hemeiron, and impurities including coexisting amino acids and ergothioneinewere removed as a precipitate to obtain a selenium concentrate (4)including 2240 μg of selenium-containing compounds. The selenium contentin the selenium concentrate (4) measured by fluorometry was 4.5 μg/ml(2240 μg/kg).

Step 3: Purification of Selenium-Containing Compound by HPLC

1) HPLC

The selenium concentrate (4) obtained by the step 2 was added to agel-filtration column (“Ultrahydrogel 120” manufactured by Waters Co.,inner diameter: 7.8 mm, column length: 300 mm) that was equilibratedwith a 0.1 M acetic acid aqueous solution including 30% of acetonitrile,passed through the column at a flow rate of 2 ml/min to isolate theselenium-containing compound, and eluted at a retention time of 4 to 8minutes (FIG. 1A).

The eluate was evaporated using a centrifugal evaporator (“VR-1”manufactured by Sakuma Seisakusho Co., Ltd.), added to a C18reversed-phase column (“Atlantis dC18 column” manufactured by WatersCo., inner diameter: 4.6 mm, column length: 250 mm), and passed throughthe column at a flow rate of 1 ml/min using 0.1% acetic acid as themobile phase. A selenium-containing component eluted when 1 to 6 minuteshad elapsed after the start of purification, was collected. The seleniumcontent in the eluate was 5000 μg/g (FIG. 1B). Ergothioneine was alsopresent in the eluate.

The eluate was added to a gel-filtration column (“Ultrahydrogel 120”manufactured by Waters Co., inner diameter: 7.8 mm, column length: 300mm) that was equilibrated with a 0.1 M acetic acid aqueous solutionincluding 30% of acetonitrile, and passed through the column at a flowrate of 2 ml/min to isolate the selenium-containing compound included inthe selenium-containing component. Selenium was directly measured byfluorometry or ICP-MS (inductively coupled plasma-mass spectrometry),and a fraction having a high selenium content was collected. Theselenium-containing compound was eluted at a retention time of 4 to 8minutes (FIG. 1C). The above chromatography operation was repeated twiceto purify the selenium-containing compound. FIG. 1 shows a case wherethe dark muscle of Thunnus obesus was used as the sample.

The selenium content was determined by fluorometry using 0.005 ml of thepurified selenium-containing compound (sample) (dark muscle of Thunnusorientalis: 5.5 μg/ml, dark muscle of Thunnus maccoyii: 1.3 μg/ml, darkmuscle of Thunnus obesus: 0.4 μg/ml, and dark muscle of Xiphias gladius:129 μg/ml).

2) ICP-MS

0.1 M ammonium formate was passed through an apparatus connected to anHPLC pump (“Pu712” manufactured by GL Sciences Inc. Ltd.), a sampleinjector (“9725i” manufactured by Rheodyne), and an ICP-massspectrometer (“ELAN DRCII” manufactured by Perkin-Elmer) at a flow rateof 0.5 to 1 ml. 2.5 μl of the HPLC fractions isolated in 1) weresequentially injected at intervals of 15 seconds while monitoringselenium-82 (natural isotope of selenium) (0.4 seconds per atomic massunit). The selenium count increased momentarily only when the sampleflowed, and immediately returned to the level before injection. Sincethe peak level increases depending on the selenium content, achromatogram of selenium purified by HPLC was obtained off-line. Afraction having a selenium peak of 1,000,000 cps or more was collected.

ICP-MS Measurement Conditions

Scans/reading: 1, readings/replicate: 800, replicates/measurement: 1,resident time per mass number: 0.4 sec

Step 4: Analysis of Chemical Structure of Selenium-Containing Compound

The selenium-containing compound obtained from the dark muscle ofThunnus orientalis and purified in the step 3 was subjected toelectrospray ionization-mass spectrometry (ESI-MS) using a protonnuclear magnetic resonance analyzer (¹H NMR), a quadrupole massspectrometer (“Quattro II” manufactured by Micromass), and a precisionmass spectrometer (“MS700” manufactured by JEOL Ltd.) to analyze thechemical structures of the main selenium-containing compounds includedin the tissues. FIG. 3 shows a 500 MHz ¹H NMR (“ECA500” manufactured byJEOL Ltd.) spectrum. Signals of saturated hydrogen were observed atδ3.08 (d, J=3.4 Hz, 2H), δ3.16 (s, 9H), and δ3.80 (dd, J=3.7, 11.2 Hz,1H) in deuterated water. These signals agree well with methylenehydrogen at position 3 (δ3.10 (m, 2H)), a methyl group (δ3.19 (s, 9H)),and methine hydrogen at position 2 (δ3.80 (dd, J=4.56, 10.98 Hz, 1H)) ofergothioneine (J. Xu and J. C. Yadan, Synthesis of L-(+)-Ergothioneine,J. Org. Chem., 60, 6296 to 6301 (1995)). The aromatic-ring hydrogen atposition 4 of the pyrimidine ring was observed at δ7.00 (s, 1H).Specifically, the signal shifted to the low-field side by 0.3 ppm fromthe value 6.70 (s, 1H) disclosed in the literature (J. Xu and J. C.Yadan, Synthesis of L-(+)-Ergothioneine, J. Org. Chem., 60, 6296 to 6301(1995)). Therefore, it was confirmed that the sulfur atom ofergothioneine had been substituted by a selenium atom.

A molecular ion peak at which one hydrogen ion was added to thestructure shown by the chemical formula 1 was observed by quadrupolemass spectrometry, and an isotope pattern characteristic of a moleculecontaining one selenium atom was observed (Table 1). A molecular ionpeak indicative of two selenium atoms was observed by accurate massanalysis around a mass number of 553.056 (Table 1). The chemical formulaestimated from the mass number was C₁₈H₂₈N₆O₄Se₂+H⁺. The molecularweight and the isotope distribution theoretically calculated from theatomic weight and the natural isotope abundance ratio of each elementagreed well with the observed values.

Table 2 shows the fragment ion composition determined by MS/MS analysis.It was estimated from the fragment ion composition that the resultingselenium-containing compound was a dimer (chemical formula 4: oxidizeddimer) having a diselenide structure including two selenium atoms, apyrimidine ring bonded to selenium, a trimethylammonium group, and acarboxyl group (see FIG. 2).

TABLE 1 Mass spectrometry of selenium-containing compound (relative peakvalue) m/z theoretical value Detected ion (ion valence) Accurate massanalysis 553.0584 553.0562 551.0594 551.0543 549.0607 549.0577 550.0620550.0588 555.0587 555.0540 552.0619 552.0627 554.0611 554.0626 547.0625547.0573 548.0630 548.0602

TABLE 2 Analysis of fragment ion by MS/MS Detected ion Molecular formula553.0562 C₁₈H₂₉N₆O₄Se₂ 233.0422 C₈H₁₅N₃Se 173.9692 C₅H₆N₂Se 79.9165 Se59.0730 C₃H₉N4. Physico-Chemical Properties of Selenium-Containing Compound

Table 3 shows property values of the above selenium-containing compound(chemical formula 4: oxidized dimer). Table 3 shows the UV absorption(absorption maximum: 260 nm) and fluorescence (maximum excitationwavelength: 240 nm, 280 nm, and 338 nm, maximum fluorescence wavelength:405 nm). A monomer was obtained by reducing the selenium-containingcompound using 10 mM dithiothreitol, and had a molecular weight ofm/z=278.0 (see Table 3). Therefore, it was confirmed that theselenium-containing compound was a dimer (chemical formula 4: oxidizeddimer) in which the individual selenium-containing compounds are bondedthrough a diselenide bond.

The monomer was a novel selenium-containing compound (chemical formula2) having a chemical structure in which the sulfur atom of the thiolgroup of ergothioneine was substituted by a selenium atom. Since thenovel selenium-containing compound had a chemical structure similar tothat of ergothioneine, the novel selenium-containing compound was named“selenoneine”.

TABLE 3 Physico-chemical properties of selenium- containing compound(chemical formula 4) Item Properties ¹H NMR (D₂O) δ3.08 (d, J = 3.4 Hz,2H), δ3.16 (s, 9H), δ3.80 (dd, J = 3.7, 11.2 Hz, 1H), δ7.00 (s, 1H)Molecular weight Monomer: m/z = 278.0, dimer: m/z = 553.06 (M + H)⁺Fluorescence Maximum excitation/fluorescence wavelength 240/405 nm(molar fluorescence coefficient: 0.823 × 10³ cm⁻¹ · mol⁻¹) 280/405 nm(molar fluorescence coefficient: 1.51 × 10³ cm⁻¹ · mol⁻¹) 337/405 nm(molar fluorescence coefficient: 1.46 × 10³ cm⁻¹ · mol⁻¹) UV absorptionMaximum absorption wavelength: 260 nm (molar fluorescence coefficient:525 cm⁻¹ · mol⁻¹) Color Yellow Solubility Soluble in water, ethanol, andmethanol; scarcely soluble in acetone and acetonitrile2) Extraction of Selenium-Containing Compound from Whale Meat

350 g of the lean meat (selenium content: 0.91 mg/kg) of Globicephalamacrorhyncus was minced to dimensions of 1×1×1 cm or less. The mincedlean meat was put into a polyethylene bottle with a lid. After theaddition of a 4-fold amount of methanol cooled to 0° C. or less, themixture was allowed to stand at 0° C. for 1 month or more. The seleniumcontent in the lean meat of Globicephala macrorhyncus was obtained bydecomposing 0.1 g of the lean meat using a mixed acid, and determiningthe selenium content by fluorometry.

1 l of the ethanol extract from which the meat had been removed wasevaporated to dryness using a rotary evaporator to obtain a seleniumconcentrate (1) including a heme iron complex of a selenium-containingcompound (chemical formula 3).

After the addition of 100 ml of a 0.1% glutathione (reduced form)aqueous solution to the selenium concentrate (1), the mixture wasstirred at room temperature for 12 hours. After the addition of 400 mlof cold ethanol, the mixture was filtered to remove heme iron. Theextract was evaporated using a rotary evaporator to obtain a seleniumconcentrate (2).

The selenium concentrate (2) was dissolved in 10 ml of cold water. Afterthe addition of 50 ml of cold ethanol, the precipitate was removed byfiltration. The extract was collected into a recovery flask, andevaporated using a rotary evaporator to obtain a selenium concentrate(3) including a selenium-containing compound (chemical formula 4:oxidized dimer).

The selenium concentrate (3) was dissolved in 10 ml of cold water. Theselenium content in the selenium concentrate (3) thus obtained from thewhale meat was measured by fluorometry. The yield of selenium was foundto be 92 μg.

3) Extraction of Selenium-Containing Compound from Tuna Blood

Extraction Example 1

The blood (selenium content: 29.8 mg/l) of Thunnus orientalis washemolyzed by adding a 5-fold amount of cold water, and centrifuged(6000×g, 10 min). The supernatant liquid was collected, and evaporatedto obtain a selenium concentrate (1). The selenium content in a driedproduct obtained by evaporating the selenium concentrate (1) to drynessusing a centrifugal evaporator was 111 mg/kg.

Extraction Example 2

A 5-fold amount of cold ethanol was added to 100 ml of the blood(selenium content: 34.7 mg/l) of Thunnus orientalis, and the solid wasground using a POLYTRON™ homogenizer. The selenium content in the bloodof Thunnus orientalis was obtained by decomposing 0.01 ml of the bloodusing a mixed acid, and determining the selenium content by fluorometry.After removing the precipitate by centrifugation (6000×g, 20 min), theremaining mixture was evaporated to dryness using a rotary evaporator toobtain a selenium concentrate (1) including a heme iron complex of aselenium-containing compound (chemical formula 3).

The selenium concentrate (1) was dissolved in 50 ml of cold water. Afterthe addition of 300 ml of cold acetonitrile (for large quantitypreparative liquid chromatography) (manufactured by Kanto Kagaku Co.,Ltd., 99.8%, cooled to 0° C. or less), the mixture was stirred, and thenallowed to stand. The upper acetonitrile layer was collected via atwo-phase separation to obtain a selenium concentrate (2) including aselenium-containing compound (chemical formula 4: oxidized dimer). Hemeiron was removed from the selenium-containing compound by extractionwith acetonitrile. The selenium content in a dried product obtained byevaporating the selenium concentrate (2) obtained from the blood ofThunnus orientalis to dryness using a centrifugal evaporator was 3100mg/kg.

Extraction Example 3

0.3 g of a dithiothreitol powder (manufactured by Nacalai Tesque, Inc.)was added to 300 ml of the blood of Thunnus orientalis aimed atisolating a selenium-containing compound from heme iron. The seleniumcontent in the blood of Thunnus orientalis was obtained by decomposing0.01 ml of the blood using a mixed acid, and determining the seleniumcontent by fluorometry.

After the addition of 300 ml of a mixed solvent of tetrahydrofuran(manufactured by Wako Pure Chemical Industries, Ltd.) anddichloromethane (manufactured by Wako Pure Chemical Industries, Ltd.)(1:1), the mixture was gently mixed. After the addition of 300 ml ofdichloromethane, the mixture was gently mixed again. The lower organicsolvent layer was collected, and evaporated using a rotary evaporator toobtain a selenium concentrate (1) including a selenium-containingcompound (chemical formula 4: oxidized dimer).

The selenium concentrate (1) was dissolved in 15 ml of cold water, andthe precipitate was removed by centrifugation (6000×g, 20 min). Theselenium content in the selenium concentrate (1) was 66 mg/l.

4) Extraction of Selenium-Containing Compound from Hepatopancreas ofThunnus orientalis

100 g of the hepatopancreas (selenium content: 8.1 mg/kg, mercurycontent: 3.9 mg/kg) of Thunnus orientalis was cut to a thickness of 5 mmor less, and put into a polyethylene bottle with a lid. After theaddition of a 2-fold amount of ethanol cooled to 0° C. or less, themixture was allowed to stand at 0° C. for 1 month or more. The ethanolextract from which the solids had been removed by filtration wasevaporated to dryness using a rotary evaporator to obtain a seleniumconcentrate (1) including a heme iron complex of a selenium-containingcompound (chemical formula 3).

The selenium content in the hepatopancreas of Thunnus orientalis wasobtained by decomposing 0.2 g of the hepatopancreas using a mixed acid,and determining the selenium content by fluorometry. The mercury contentwas determined by cold vapor atomic absorption spectrometry.

The selenium concentrate (1) was dissolved in 20 ml of cold water. Afterthe addition of 200 ml of cold acetonitrile (manufactured by Wako PureChemical Industries, Ltd.), the mixture was stirred, and then allowed tostand. The upper acetonitrile layer was collected via a two-phaseseparation to obtain a selenium concentrate (2) including aselenium-containing compound (chemical formula 4: oxidized dimer). Hemeiron was removed from the selenium-containing compound by extractionwith acetonitrile. The selenium concentrate (2) thus obtained from thehepatopancreas of Thunnus orientalis was evaporated using a rotaryevaporator, and dissolved in a small quantity of water to obtain 1 ml ofa crude selenium extract.

The crude selenium extract was wet-decomposed using a mixed acid, andthe selenium content (fluorometry), the iron content (ICP emissionspectrometry), the mercury content (cold vapor atomic absorptionspectrometry), and the nitrogen content (Kjeldahl method) were analyzed.The acetonitrile soluble extract had a selenium content of 50 mg/kg, aniron content of 1.8 mg/kg, a mercury content of 0.012 mg/kg, and anitrogen content of 45,900 mg/kg. The acetonitrile insoluble extract hada selenium content of 12 mg/kg, an iron content of 103 mg/kg, a mercurycontent of 0.001 mg/kg, and a nitrogen content of 168,000 mg/kg.

It was thus confirmed that the mercury content decreases to 1/300th ofthat of the hepatopancreas (i.e., an organ having a mercury contentseveral times that of muscle) by extraction of selenium. Since theselenium concentrate contains a large amount of nitrogen and iron (i.e.,nutrient elements), the selenium concentrate can be safely andeffectively used as a feed additive and the like.

5) Extraction of Selenium-Containing Compound from Dark Muscle ofThunnus maccoyii

500 ml of cold water was added to 100 g of the dark muscle of Thunnusmaccoyii, and the solid was ground using a POLYTRON™ homogenizer. Theprecipitate was removed by centrifugation (6000×g, 20 min) to obtain awater extract (1).

After the addition of 2-mercaptoethanol (final concentration: 10 mM) ordithiothreitol (final concentration: 50 mM) to 100 ml of the waterextract (1), the mixture was stored at room temperature for 1 hour(reduction process) to obtain an extract (2). 20 ml of the water extract(1) was heated in a boiling water bath to obtain a hot water extract(2). The pH of the water extract (1) was adjusted to 3 by adding 1 Mhydrochloric acid, and allowed to stand at room temperature for 10minutes to obtain an acid extract (2).

After the addition of 400 ml of cold ethanol to each extract (2), solidswere removed by centrifugation (6000×g, 20 min). The residue wasevaporated using a rotary evaporator to obtain a selenium concentrate(1). After the addition of acetonitrile to the selenium concentrate (1),the mixture was stirred, and then allowed to stand. The upperacetonitrile layer was collected via a two-phase separation, evaporatedusing a rotary evaporator, and dissolved in a small quantity of water toobtain a crude selenium extract including a selenium-containing compound(chemical formula 4: oxidized dimer).

The crude selenium extract was wet-decomposed using a mixed acid, andthe selenium content (fluorometry), the iron content (ICP emissionspectrometry), and the mercury content (cold vapor atomic absorptionspectrometry) were analyzed. Table 4 shows the selenium content, theiron content, and the mercury content in each crude selenium extract.

TABLE 4 Se (μg) Fe (μg) Hg (μg) No additive 3.8 14 0.009 DTT (50 mM) 4.7201 0.700 Mercaptoethanol (10 mM) 1.4 15 0.032 Hot water 2.4 33 <0.001Hydrochloric acid (pH: 3.0) 5.0 21 0.125

Example 2 Production of Selenol-Type Selenium-Containing Compound orSelenoketone-Type Selenium-Containing Compound

The selenoketone-type selenium-containing compound of Example 1(chemical formula 3) was subjected to selenoketone-selenol tautomericconversion to obtain a selenol-type selenium-containing compound(chemical formula 2).

The selenium-containing compound according to one embodiment of theinvention has a molecular structure in which a selenol group is bondedto a carbon atom at position 2 of a pyrimidine ring. A selenol groupforms a tautomer that is in an equilibrium state with a selenoketonegroup. The selenoketone group and the selenol group are functionalgroups that are unstable to heat and oxygen. The selenoketone groupliberates a low-molecular-weight selenium compound, and rapidly losesselenium. On the other hand, the selenol group easily produces arelatively stable dimer (oxidized dimer) in a gas phase or a non-polarsolvent (e.g., cyclohexane or tetrahydrofuran) in which the equilibriumshifts to the selenol isomer (J. Elguero, C. Marzin, A. R. Katritzky,and P. Linda, “The Tautomerism of Heterocycles”, A. R. Katritzky and A.J. Boulton Eds.; Advances in Heterocyclic Chemistry, Supplement No. 1;Academic Press: New York, 1976).

A 2-fold amount of a mixed solvent of tetrahydrofuran and cyclohexane(1:1) was added to a selenium concentrate including aselenium-containing compound (chemical formula 3), and the mixture wasstirred at room temperature overnight to obtain an emulsion. Theemulsion was evaporated using a rotary evaporator. After extracting theselenium-containing compound with purified water, water was removedusing a centrifugal evaporator to collect a selenium concentrate (5)including a yellow oily selenol-type selenium-containing compound(chemical formula 2). The selenium content in the selenium concentrate(5) was 2400 mg/kg.

Example 3 Optimization of Selenium-Containing Compound ExtractionConditions

1. Study of Extraction Solvent

The selenium-containing compound extraction conditions were optimized byextracting a selenium-containing compound using a different extractionsolvent, and comparing the yields of the selenium-containing compound.The internal organ of Beryx splendens was used as the sample, andethanol, methanol, or acetone was used as the extraction solvent.

80% cold ethanol, cold methanol (cooled to −40° C. or less), or acetonewas added to 20 g of the internal organ of Beryx splendens as theextraction solvent, and the solid was ground using a POLYTRON™homogenizer. After removing the precipitate by centrifugation (6000×g,20 min), the mixture was evaporated using a rotary evaporator to obtaina selenium concentrate (1) including a heme iron complex of aselenium-containing compound (chemical formula 3).

After the addition of 200 ml of acetonitrile to the selenium concentrate(1), the mixture was stirred, and then allowed to stand. The upperacetonitrile layer was collected via a two-phase separation to obtain aselenium concentrate (2) including a selenium-containing compound(chemical formula 4: oxidized dimer). The selenium concentrate (2) thusobtained was evaporated using a rotary evaporator, and dissolved in asmall quantity of water to obtain 12 ml of a methanol extract, 14 ml ofa 80% ethanol extract, or 8 ml of an acetone extract (i.e., crudeselenium extract).

The crude selenium extract was wet-decomposed using a mixed acid, andthe selenium content, the iron content (ICP emission spectrometry), andthe mercury content (cold vapor atomic absorption spectrometry) wereanalyzed.

When using methanol as the extraction solvent, the selenium content andthe mercury content in the extract were 8.9 mg/l and 0.046 mg/l,respectively. When using 80% ethanol as the extraction solvent, theselenium content and the mercury content in the extract were 4.5 mg/land 0.008 mg/l, respectively. When using acetone as the extractionsolvent, the selenium content and the mercury content in the extractwere 5.6 mg/l and 0.010 mg/l, respectively. It was thus confirmed that aselenium concentrate including a selenium-containing compound at a highconcentration can be obtained using each of the above extractionsolvents.

2. Purification of Selenium-Containing Compound Using Ion-Exchange ResinColumn

The purity of a selenium concentrate including a selenium-containingcompound was increased by removing impurities by purification using anion-exchange resin column.

Specifically, 100 g of the dark muscle of Thunnus orientalis was mincedto dimensions of 1×1×1 cm or less. After the addition of a 10-foldamount of cold ethanol, the dark muscle was ground using a POLYTRON™homogenizer, and centrifuged (6000×g, 20 min). The supernatant liquidwas collected, and evaporated using a rotary evaporator to obtain aselenium concentrate (1) including a heme iron complex of aselenium-containing compound (chemical formula 3).

The selenium concentrate (1) was dissolved in 100 ml of cold methanol,and filtered. The extract was evaporated using a rotary evaporator toobtain a selenium concentrate (2). Cold water was added to the seleniumconcentrate (2) so that the liquid volume was 30 ml.

The selenium concentrate (2) to which cold water was added, was added toa Sep-Pack Accell Plus QMA anion-exchange column (manufactured by WatersCo., internal volume: 50 mg) that was equilibrated with water. Afterwashing the column with 20 ml of water, 10 ml of a 0.1% acetic acidaqueous solution was added to the column to elute a selenium concentrate(3) including a selenium-containing compound. The selenium content inthe selenium concentrate (3) including a selenium-containing compoundwas 5.8 μg/ml.

Example 4 Production and Analysis (Mass Spectrometry) of Analog ofSelenium-Containing Compound

In order to analyze the chemical structure and the reactivity of aselenium-containing compound produced in vivo and in vitro, theselenium-containing compound (chemical formula 4: oxidized dimer)included in the selenium concentrate (extracted from the dark muscle ofThunnus orientalis) prepared in Example 1 was reduced andcarboxymethylated (Masatsune Ishiguro, Chemical modification of SHgroup, Biochemistry Experimental Method 8, Japan Scientific SocietiesPress (1978)) to obtain a chemically modified reaction product (analogof selenium-containing compound). The reaction product was identified bymass spectrometry.

Specifically, the selenium-containing compound was reduced usingdithiothreitol in accordance with the cystine reduction andcarboxymethylation method, followed by the addition of iodoacetic acidto obtain a reaction product. The reaction product was purified using agel-filtration column (“Ultrahydrogel 120” manufactured by Waters Co.,inner diameter: 7.8 mm, column length: 300 mm) that was equilibratedwith 1 M ammonium acetate including 30% of acetonitrile to obtain acomponent having UV absorption at 260 nm as a chemically modifiedreaction product (analog of selenium-containing compound).

The chemically modified reaction product (analog of selenium-containingcompound) was analyzed (mass spectrometry) within the mass range (m/z)of 100 to 600 using an ESI-MS (“Quattro II” manufactured by Micromass).When analyzing the mass of the product using the ESI-MS, the molecularions of the oxidized dimer of the selenium-containing compound (m/z=553,277) and an ergothioneine addition product (m/z=491) were observed, anda product in which the selenol group of the selenium-containing compoundmonomer was tarboxymethylated (ions: m/z=233 (277-CO₂) and m/z=174(277-CO₂—N(CH₃)₃)) was detected (Table 5 and FIG. 4).

The selenium-containing compound (chemical formula 4: oxidized dimer)was reduced at room temperature for 30 minutes using 10 mMdithiothreitol, and the mass of the product was analyzed using theESI-MS. A reduced monomer (chemical formula 2) was detected.

It is known that a plurality of selenium isotopes exist. The theoreticalmass number of the M+H⁺ ion of the reduced monomer of theselenium-containing compound based on the selenium isotope abundanceratio is 278.0 (100.0%), 276.0 (49.5%), 274 (18.8%), 280 (18.5%), 275.0(17.4%), 279.0 (11.1%), 277.0 (5.4%), 281.0 (2.0%), and 272 (1.8%) (thepercentage in the parentheses is the theoretical abundance ratio of eachion), and the measured value was 278.0 (100.0%), 276.1 (51.8%), 274(18.9%), 280 (18.3%), 275.1 (18.9%), 279.1 (11.6%), 277.1 (7.3%), 281.0(2.4%), and 272 (2.7%). The molecular weight theoretically calculatedfrom the atomic weight and the natural isotope abundance ratio of eachelement agrees well with the measured values, and a signal distributionin which one molecule contains one selenium atom was obtained.

It was thus confirmed that the main selenium-containing compoundsincluded in the tissues of fish are selenium-containing compound dimers(chemical formula 4: oxidized dimer) in which the individualselenium-containing compounds are bonded through a diselenide bond.

TABLE 5 Identification of analog by chemical modification ofselenium-containing compound m/z theoretical value (ion valence)Detected ion Chemical formula 553.1, 551.1, 549.1 (1+ ion), 553.1,551.1, 549.1, 277.25 Selenoneine dimer 277 (2+ ion) 505.1, 503.1, 506.1,507.1 505.4, 503.4, 506.4, 507.4 Ergothioneine addition product 336.0,334.0, 332.0, 338.0 336.2, 334.2, 332.3, 338.3 Reduced carboxymethylatedproduct 280.0, 278.0, 276.0, 275.0, 274.0 280.1, 278.0, 276.1, 275.1,274.1 Reduced monomer

Example 5 Use of Selenium-Containing Compound

The applicability of a selenium-containing compound (chemical formula 4:oxidized dimer) purified in the same manner as in Example 1 as aquantitative analysis standard substance or a medium additive, anantioxidant, a metmyoglobin formation inhibitor, and the like wasdetermined

1. Utility of Selenium-Containing Compound as Standard Substance

Whether or not a selenium-containing compound obtained according to theinvention can be used as a standard substance for quantitativelyanalyzing a selenium-containing compound in a sample was determined.

Specifically, the selenium-containing compound (chemical formula 4:oxidized dimer) according to one embodiment of the invention,glutathione peroxidase (SIGMA), a selenium standard solution (seleniumdioxide aqueous solution (CAS No. 7446-08-4, manufactured by Wako PureChemical Industries, Ltd.), selenocystine (manufactured by SIGMA), orselenomethionine (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was usedas the sample, and introduced into an ICP-MS by flow injection or HPLCusing a neutral buffer as the mobile phase. Organic selenium included inthe sample was detected as a selenium-82 ion by ionization due to argonplasma excitation.

The selenium-82 ion was measured by dissolving the selenium-containingcompound (chemical formula 4: oxidized dimer) according to oneembodiment of the invention in 0.1 M ammonium formate, and introducingthe solution into an inductively coupled plasma mass spectrometer.Glutathione peroxidase, sodium selenite (manufactured by Wako PureChemical Industries, Ltd.), selenocystine, and selenomethionine wereprepared so that the selenium content was 100 pg/μl. 5 μl, 10 μl, or 20μl of each sample was introduced, and a signal of selenium-82 wasdetected using an ICP-MS (“ELAN DRCII” manufactured by Perkin-Elmer).The concentration of the selenium-containing sample was corrected usingthe selenium content analyzed by fluorometry.

As shown in FIG. 5, the selenium-containing compound (chemical formula4: oxidized dimer) according to one embodiment of the invention and theselenium-containing samples (1: glutathione peroxidase, 2: seleniumdioxide aqueous solution, 3: L-selenocystine, 4: L-selenomethionine)were detected at different retention times, and a calibration curve witha correlation coefficient of 0.99 or more was obtained. It was thusconfirmed that these substances can be isolated and quantitativelydetermined (Table 6).

TABLE 6 Analysis of selenium-containing compound and selenium-containingsamples by ICP-MS Elution time Peak area (min) (Se pmol⁻¹) Glutathioneperoxidase 5.4 3492 Selenium dioxide aqueous solution 7.4 5006Selenocystine 7.8 2954 Selenomethionine 9.8 2789 Selenium-containingcompound 10.5 3480 (chemical formula 4)2) Use of Selenium-Containing Compound (Chemical Formula 4: OxidizedDimer) as Standard Substance

A selenium-containing compound included in the dark muscle of Thunnusorientalis, the ordinary muscle of Thunnus orientalis, the blood ofThunnus orientalis, the spleen of Thunnus orientalis, the liver ofThunnus orientalis, the gills of Thunnus orientalis, the heart of achicken, the liver of a chicken, the hepatopancreas of Todarodespacificus, and human blood was quantitatively analyzed in the samemanner as in 1) using the selenium-containing compound (chemical formula4: oxidized dimer) according to one embodiment of the invention as thestandard substance.

Specifically, 0.1 g of the dark muscle of Thunnus orientalis, theordinary muscle of Thunnus orientalis, the blood of Thunnus orientalis,the spleen of Thunnus orientalis, the liver of Thunnus orientalis, thegill of Thunnus orientalis, the heart of a chicken, the liver of achicken, the hepatopancreas of Todarodes pacificus, or human blood wasground using a POLYTRON™ homogenizer (manufactured by Kinematica, Inc.)together with 0.5 ml of Millipore water, and centrifuged at 12,000 rpmfor 5 minutes. The supernatant liquid was used as the sample. The samplewas filtered (syringe filter No. 6872-1304 manufactured by Whatman,Inc.), 2 to 10-fold diluted with an HPLC mobile phase, and introducedinto an HPLC-ICP-MS. A selenium-containing compound contained in thesample was quantitatively analyzed under measurement conditions shown inTable 7.

As shown in FIG. 6 and Table 8, it was found that the dark muscle ofThunnus orientalis includes the selenium-containing compound(selenium-containing compound having a skeleton shown by the chemicalformula 1, hereinafter referred to as “(chemical formula 1)”) accordingto one embodiment of the invention in an amount corresponding to aselenium content of 95%. Specifically, the content of theselenium-containing compound (chemical formula 1) according to oneembodiment of the invention was indirectly estimated by indicating thecontent of selenium-containing compounds other than theselenium-containing compound (chemical formula 1) according to oneembodiment of the invention (i.e., compounds that are eluted at aretention time differing from that of the selenium-containing compound(chemical formula 1) according to one embodiment of the invention, andinclude selenium) with respect to the total selenium content by the arearatio of the peak of selenium-containing compounds other than theselenium-containing compound (chemical formula 1) according to oneembodiment of the invention to the total selenium peak.

For example, the ratio of the selenium-containing compound (chemicalformula 1) according to one embodiment of the invention included in thedark muscle of Thunnus orientalis with respect to the total seleniumcontent was calculated as follows.(100−2.5)*2018.9/(2018.9+44.7)=95.3%

A selenium-containing protein (i.e., a polymer other than selenoproteinP that has a molecular weight of 20,000 or more and contains oneselenium atom in the molecule), the selenium-containing compound(chemical formula 1) according to one embodiment of the invention, andselenium-containing compounds other than the selenium-containingcompound (chemical formula 1) according to one embodiment of theinvention were detected from various tissues. The selenium-containingprotein content is indicated by the GPX equivalent amount calculatedfrom Table 7, and the content of selenium-containing compounds otherthan the selenium-containing compound (chemical formula 1) according toone embodiment of the invention is indicated by the ratio of the area tothe detected total peak area.

As shown in FIG. 7 and Table 9, it was confirmed that theselenium-containing compound (chemical formula 1) according to oneembodiment of the invention was included (distributed) in the ordinarymuscle of Thunnus orientalis, the blood of Thunnus orientalis, the heartof a chicken, the liver of a chicken, the hepatopancreas of Todarodespacificus, and human blood in the same manner as in the dark muscle ofThunnus orientalis.

The dark muscle and the ordinary muscle of Thunnus orientalis werefractionated into a cytoplasmic fraction (78000×g, supernatant liquid),a microsomal fraction (precipitated at 22500×g to 78000×g), and amitochondrial fraction (precipitated at 700×g to 8000×g) bycentrifugation (Michiaki Yamashita, Studies on cathepsins in the muscleof chum salmon, Bulletin of the National Research Institute of FisheriesScience, 5, 9-114 (1993)), and localization of the selenium-containingcompound (chemical formula 1) according to one embodiment of theinvention in the cells was analyzed by the method described in Example3. It was confirmed that the selenium-containing compound (chemicalformula 1) according to one embodiment of the invention was localized incytoplasm together with glutathione peroxidase (i.e.,selenium-containing protein) (Table 9). It is considered that theselenium-containing compound (chemical formula 1) according to oneembodiment of the invention is involved in an in vivo antioxidant effectin the cells.

TABLE 7 HPLC-ICP-MS analysis conditions Element Se RF output (kW) 1.5Nebulizer gas flow rate (Ar L/min) 1 Auxiliary gas flow rate (Ar L/min)1.3 Plasma gas flow rate (Ar L/min) 17 Makeup gas flow rate 0 Lensvoltage (V) 8.2 Reaction gas None Mass number (amu) 82 Pulse stagevoltage 1050 RPq 0.25 HPLC conditions Column Ultrahydrogel 120 (7.8 ×300 mm) Mobile phase 0.1M ammonium formate (pH: 7.0) Flow rate 1.0ml/min

TABLE 8 Selenium content in living tissues Ratio (%) ofselenium-containing compounds other than Selenium-containingselenium-containing compound Selenium-containing compound (chemical(chemical formula 1) with respect to Species/tissue protein (nmol/g)formula 1) (nmol/g) total selenium content Thunnus orientalis dark 44.72018.9 2.5 muscle Thunnus orientalis 13.1 26.8 11.6 ordinary muscleThunnus orientalis blood 23.6 609 0.0 Chicken heart 20.2 1.1 44 Chickenliver 66.7 3.0 26 Chicken gizzard 26.8 0.0 39 Pig liver 80.0 0.0 38Todarodes pacificus 50.3 93.1 21 hepatopancreas

TABLE 9 Intracellular localization of novel selenium-containing compoundRatio (%) of selenium-containing compounds other thanSelenium-containing selenium-containing compound Species/tissueSelenium-containing compound (chemical (chemical formula 1) with respectto Cell fraction protein (nmol/ml) formula 1) (nmol/ml) total seleniumcontent Thunnus orientalis dark muscle Cytoplasmic fraction 17.34 28.870.01 Microsome fraction 2.41 0.35 0.01 Mitochondrial fraction 0.40 0.200.02 Thunnus orientalis ordinary muscle Cytoplasmic fraction 0.71 1.040.00 Microsome fraction 0.11 0.16 0.02 Mitochondrial fraction 0.10 0.160.042. Chemical Antioxidant Ability of Selenium-Containing Compound

In order to evaluate the chemical antioxidant ability of theselenium-containing compound (chemical formula 4: oxidized dimer)according to one embodiment of the invention, the1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging ability wasmeasured (Hideo Kimura, Kota Naganuma, Masato Kojima, Masakazu Komatsu,Takumi Onda, and Masao Tsuji, Yamanashi Prefectural IndustrialTechnology Center Report, No. 22, pp. 59-63 (2008)).

Specifically, a selenium-containing compound (selenium content: 1.62 μM)derived from the meat of Xiphias gladius, a 10 mM aqueous solution of awater-soluble vitamin E derivative (commercially available antioxidant)(Trolox (registered trademark) (CAS No.: 56305-04-5, manufactured byRoche)), and a 4.36 mM aqueous solution of (+)-ergothioneine (CAS No.:497-30-3, manufactured by SIGMA) were repeatedly 2-fold diluted withpurified water to prepare 2 to 128-fold diluted test solutions.

10 μl of the test solution and 100 μl of a DPPH solution (0.35 mM,prepared by dissolving 1.38 mg of DPPH in 5 ml of ethanol, and adding 5ml of a 0.1 M phosphate buffer (pH: 6.8) to the solution) were mixed ona 96-well micro test plate (BD Falcon (registered trademark), #1722,manufactured by Becton, and Dickinson and Company), and reacted at roomtemperature for 30 minutes in a dark place. The absorbance at 595 nm wasthen measured. A case of using purified water (absorbance: 0.59) as thetest solution was determined that the DPPH radical-scavenging rate was0%, and a case of using 10 mM Trolox (registered trademark) (absorbance:0.19) as the test solution was determined that the DPPHradical-scavenging rate was 100%. The concentration at which the DPPHradical-scavenging rate was 50% was determined to be the RS₅₀ of eachsubstance, and taken as the antioxidant ability.

It was confirmed that the selenium-containing compound (chemical formula4: oxidized dimer) according to one embodiment of the invention has aradical-scavenging ability equal to that of the water-soluble vitamin Ederivative (Trolox (registered trademark)) at a concentration 1/456th ofthat of the water-soluble vitamin E derivative (Trolox (registeredtrademark)), and has a radical-scavenging ability equal to that ofL-(+)-ergothioneine (sulfur-containing antioxidant) at a concentration1/907th of that of L-(+)-ergothioneine. Specifically, it was confirmedthat the selenium-containing compound (chemical formula 4: oxidizeddimer) according to one embodiment of the invention has a very strongantioxidant ability.

TABLE 10 DPPH radical-scavenging ability of selenium-containing compoundRS₅₀ (μM) Water-soluble vitamin E derivative 880 (Trolox (registeredtrademark)) L-(+)-ergothioneine 1700 Selenium-containing compound 1.93. In Vivo Antioxidant Effect of Selenium-Containing Compound

In order to clarify the intracellular function of theselenium-containing compound (chemical formula 4: oxidized dimer)according to one embodiment of the invention, the selenium-containingcompound (chemical formula 4: oxidized dimer) according to oneembodiment of the invention was administered to human vessel endothelialcells (human umbilical cord vein HUV-EC-C, Health Science ResearchResources Bank (http://www.jhsf.or.jp)).

Specifically, human vessel endothelial cells (10,000 cells/ml) werecultured on a 48-well plate using an MCDB131 medium (manufactured byInvitrogen) containing 10% fetal bovine serum. The selenium-containingcompound was added to the culture solution so that the finalconcentration was 1, 2.5, 5, or 10 mM (control: the selenium-containingcompound was not added), and the cells were cultured at 37° C. for 24hours under 5% carbon dioxide. The passage number was set to 5 or lessin order to maintain the properties of the human vessel endothelialcells.

The mitochondrial respiratory chain activity was determined using an MTTassay kit (manufactured by Nacalai Tesque, Inc.). Specifically, afterculturing the human vessel endothelial cells, an MTT solution was addedto the culture solution in an amount 1/10th of that of the culturesolution, and the cells were cultured at 37° C. for 3 hours under 5%carbon dioxide. An equal amount of isopropanol was then added to theculture solution to extract a formazan dye, and the absorbance at 595 nmwas determined.

As shown in FIG. 8, the selenium-containing compound (chemical formula4: oxidized dimer) according to one embodiment of the invention showed acell proliferation-promoting effect at a concentration of 5 mM. Whenadding sodium selenite (manufactured by SIGMA), selenocystine(manufactured by SIGMA), or selenomethionine (manufactured by TokyoKasei Kogyo Co., Ltd.) to the culture medium of the human vesselendothelial cells to a concentration of 5 nM, cell death occurredinstead of cell proliferation. It was thus confirmed that theselenium-containing compound (chemical formula 4: oxidized dimer)according to one embodiment of the invention has very low cytotoxicityas compared with known selenium-containing compounds such as seleniousacid, selenocystine, and selenomethionine.

The in vivo antioxidant effect of the selenium-containing compound(chemical formula 4: oxidized dimer) according to one embodiment of theinvention was determined using human vessel endothelial cells.Specifically, human vessel endothelial cells (10,000 cells/ml) werecultured on a 48-well plate using an MCDB131 medium containing 10% fetalbovine serum. The selenium-containing compound was added to the culturesolution so that the final concentration was 5 mM (control: theselenium-containing compound was not added), and the cells were culturedat 37° C. for 1 hour under 5% carbon dioxide. After the addition ofhydrogen peroxide to a final concentration of 25 or 50 μM, the cellswere cultured at 37° C. for 24 hours under 5% carbon dioxide. The cellproliferation rate was then determined by the MTT assay. It was foundthat the cellular proliferative potential was improved under oxidativestress conditions due to hydrogen peroxide by adding theselenium-containing compound (chemical formula 4: oxidized dimer)according to one embodiment of the invention. Specifically, it wasconfirmed that the selenium-containing compound (chemical formula 4:oxidized dimer) according to one embodiment of the invention has an invivo antioxidant effect.

It was thus confirmed that the selenium-containing compound (chemicalformula 4: oxidized dimer) according to one embodiment of the inventionadded to a medium is incorporated in human cells, and exhibits an invivo antioxidant effect on the cells. It is considered that theselenium-containing compound (chemical formula 4: oxidized dimer)according to one embodiment of the invention functions as a reducingagent in cytoplasm.

4. Cell Proliferation-Promoting Effect of Selenium-Containing Compound

The cell proliferation-promoting effect of the selenium-containingcompound (chemical formula 4: oxidized dimer) according to oneembodiment of the invention on cultured cells was determined

Specifically, Danio rerio embryo ZE cells (National Research Instituteof Fisheries Science), Seriola quinqueradiata tail fin YT cells(National Research Institute of Fisheries Science), and Oncorhynchusmykiss gonad RTG-2 cells (ATCCCCL55) were cultured in an L-15 mediumcontaining 2% fetal bovine serum (30,000 cells/ml).

Human vessel endothelial cells (human umbilical cord vein HUV-EC-C,Health Science Research Resources Bank (http://www.jhsf.or.jp)) werecultured in an MCDB131 medium containing 10% fetal bovine serum (10,000cells/ml).

AT cells derived from the skin of a human patient with ataxiatelangiectasia syndrome (AT2KY, Health Science Research Resources Bank,JCRB0316), human skin TIG-S3 cells (Health Science Research ResourcesBank, JCRB0544), and human kidney HEK293 cells (Health Science ResearchResources Bank, JCRB9068) were cultured in a DMEM medium containing 10%fetal bovine serum (15,000 cells/ml).

The selenium-containing compound was added to each culture medium(containing 2% serum) so that the final concentration was 0, 5, 10, 25,50, or 100 nM. The Danio rerio embryo ZE cells were cultured at 25° C.,the Seriola quinqueradiata tail fin YT cells were cultured at 20° C.,and Oncorhynchus mykiss gonad RTG-2 cells were cultured at 15° C., andthe human vessel endothelial cells, the AT cells derived from the skinof a human patient with ataxia telangiectasia syndrome, the human skinTIG-S3 cells, and the human kidney HEK293 cells were cultured at 37° C.The cell count was determined by trypan blue staining after culturingthe cells for 0, 1, 2, 3, 4, and 5 days.

As shown in FIG. 9, it was confirmed that the concentration of theselenium containing compound necessary for promoting cell proliferationdiffers depending on the type of cell line. It was thus found that theselenium-containing compound significantly promotes proliferation ofanimal cells, and the amount of selenium-containing compound required topromote cell proliferation differs depending on the type of cells. Itwas thus confirmed that the selenium-containing compound can be used asa medium additive when culturing animal cells.

Since the AT cells derived from the skin of a human patient with ataxiatelangiectasia syndrome lack an ATM kinase gene that is an uppermoststream signalling factor that responds to DNA damage, aging and cancerare induced by abnormalities in DNA repair and apoptosis (K. Naka, A.Tachibana, K. Ikeda, and N. Motoyama, J. Biol. Chem., 279, pp. 2030-2037(2004)). Since the selenium-containing compound (chemical formula 4:oxidized dimer) according to one embodiment of the invention had aneffect of promoting proliferation of the AT cells, it is considered thatthe selenium-containing compound has an in vivo antioxidant effect thatrecovers the DNA damage response.

5. Metmyoglobin Formation Inhibitory Effect of Selenium-ContainingCompound

Preserved rabbit blood (Nippon Biotest Laboratories Inc.) was washedthree times with a physiological saline solution (centrifuged at 2000rpm and 4° C. for 5 min) to collect red cells. The red cells weresuspended in a 4-fold amount of an RPMI medium (GIBCO (registeredtrademark) RPMI Medium 1640 manufactured by Invitrogen Co.), andcultured at 37° C. for 1 day using a carbon dioxide incubator. Theselenium-containing compound was added to the culture solution so thatthe final concentration of the selenium-containing compound in themedium was 10, 25, or 50 nM (control: the selenium-containing compoundwas not added), and the cells were cultured. The red cells were thencollected by centrifugation (2000×g, 4° C., 5 min), washed three timeswith a phosphate buffered saline (PBS), and collected by centrifugation.The red cells were hemolyzed by adding a 10 mM sodium phosphate buffer(pH: 7.2) to the red cell pellets, and centrifuged (15,000×g, 4° C., 10min). The supernatant hemoglobin solution was then collected. Thehemoglobin solution was diluted with a 100-fold amount of a 50 mM sodiumphosphate buffer (pH: 6.8), and incubated at 37° C. for 3 hours. Theabsorbance ratio at 405 nm and 430 nm before and after incubation wasmeasured, and the rate of metmyoglobin formation of the hemoglobin wascalculated.

The active oxygen level in the red cells was also measured usinghydroxyphenyl fluorescein (HPF) (manufactured by Sekisui Medical Co.,Ltd.). The hemoglobin solution was mixed with a 100-fold amount of HPFdiluted with a 1000-fold amount of a phosphate buffered saline (PBS),and reacted at room temperature for 10 minutes. The fluorescenceintensity (excitation wavelength: 490 nm, fluorescence wavelength: 515nm) due to the reaction product with active oxygen was measured, andused as an index of the active oxygen level.

As shown in FIG. 10, the rate of metmyoglobin formation of the red cellsat each concentration of the selenium-containing compound was lower thanthat of the control. As shown in FIG. 11, the active oxygen level in thered cells achieved when adding the selenium-containing compound waslower than that of the control. It was thus confirmed that theselenium-containing compound has an in vivo antioxidant effect thatsuppresses in vivo methemoprotein formation. Specifically, it wasconfirmed that the selenium-containing compound is incorporated in cellsfrom a medium, prevents production of active oxygen, and functions as aheme iron auto-oxidation inhibitor. It was thus confirmed that theselenium-containing compound may be used for blood/red cell/internalorgan preservation technology (i.e., may be used as a metmyoglobinformation inhibitor for heme proteins (e.g., hemoglobin and myoglobin),artificial blood, transfusion preparations, or tissue preservationsolutions).

6. Heme Iron Binding Effect of Selenium-Containing Compound

Since it was confirmed that the selenium-containing compound has a hemeprotein auto-oxidation inhibitory effect (see “5. Metmyoglobin formationinhibitory effect of selenium-containing compound”), the bindingcapability of the selenium-containing compound with heme proteins wasdetermined. Physeter macrocephalus (sperm whale) myoglobin availablefrom SIGMA was used. 100 g of the dark muscle of Thunnus orientalis wasmixed with a 5-fold amount of cold water, ground using a POLYTRON™homogenizer, and centrifuged (6000×g, 10 min). The supernatant liquidwas collected, and passed through a Sephacryl-100 column (16 mm×60 cm,manufactured by Pharmacia) that was equilibrated with a 0.05 M Tris-HClbuffer (pH: 7) containing 0.1 M salt to purify myoglobin derived fromthe dark muscle of Thunnus orientalis. Hemoglobin was obtained bypurifying a lysate of the red cells of rabbit or Thunnus orientalisusing the Sephacryl-100 column.

The selenium content in the Physeter macrocephalus myoglobin, theThunnus orientalis myoglobin, the rabbit hemoglobin, and the Thunnusorientalis hemoglobin was measured. Selenium was detected from eachsample. The elements contained in the Physeter macrocephalus myoglobinwere measured by HPLC-ICP-MS. As shown in FIG. 12, iron and seleniumwere detected from the Physeter macrocephalus myoglobin.

The sample was wet-decomposed using nitric acid and hydrogen peroxide.Iron was quantitatively determined using an inductively coupled plasmaatomic emission spectrometer (ICP-AES) (“ICPE-9000” manufactured byShimadzu Corporation), and selenium was quantitatively determined byfluorometry. The Se/Fe molar ratio of the Thunnus orientalis myoglobin,the rabbit hemoglobin, and the Thunnus orientalis hemoglobin was 0.003,0.0003, and 0.001, respectively. It was thus confirmed that theselenium-containing compound (chemical formula 4: oxidized dimer)according to one embodiment of the invention was bound to a hemeprotein. It is considered that the selenium-containing compound iscoordinated to heme iron to suppress auto-oxidation of heme iron.

Since the selenium-containing compound according to one embodiment ofthe invention has an in vivo antioxidant effect, the selenium-containingcompound may be used for, or as, a drug, a functional food, anutritional supplement, a food additive, a feed additive, a mediumadditive, or an antioxidant. It is also possible to provide a method ofproducing a safe selenium-containing compound by removing heavy metalsfrom fishery processing residues (e.g., dark muscle, internal organs,and blood). The chemical form of selenium in a living body, food, andthe like can be evaluated by utilizing the selenium-containing compoundaccording to one embodiment of the invention as a standard substance.Since the selenium-containing compound according to one embodiment ofthe invention includes a selenol group having high reactivity, and hasfluorescent and UV absorption characteristics, the selenium-containingcompound may be used as unique selenium-containing chemical materials(e.g., chemical modifier, fluorescent substance, or UV absorber).

What is claimed is:
 1. A method of producing a selenium-containingcompound of chemical formula 1:

wherein R is absent, or represents hydrogen or an organic compound, themethod comprising (a) extracting the selenium-containing compound ofchemical formula 1 from a sample using an organic solvent or water, and(b) reducing the sample that includes the selenium-containing compound,or a selenium concentrate that includes the selenium-containingcompound, using a reducing agent.
 2. The method according to claim 1,comprising extracting the selenium-containing compound from the sampleusing a hydrophilic organic solvent, followed by extraction with asolvent including a hydrophobic or Panic solvent.
 3. The methodaccording to claim 2, wherein the hydrophilic organic solvent is atleast one compound selected from ethanol, methanol, acetone, andacetonitrile.
 4. The method according to claim 3, wherein thehydrophobic organic solvent is at least one compound selected fromdiethyl ether, tetrahydrofuran, cyclohexane, and dichloromethane.
 5. Themethod according to claim 1, wherein the reducing agent is a thiolreducing agent.
 6. The method according to claim 2, wherein the reducingagent is a thiol reducing agent.
 7. The method according to claim 3,wherein the reducing agent is a thiol reducing agent.
 8. The methodaccording to claim 4, wherein the reducing agent is a thiol reducingagent.
 9. The method according to claim 1, further comprisingconcentrating a selenium concentrate using an ion-exchange resin. 10.The method according to claim 1, further comprising purifying a seleniumconcentrate by high performance liquid chromatography (HPLC).
 11. Themethod according to claim 2, wherein the hydrophobic organic solvent isat least one compound selected from diethyl ether, tetrahydrofuran,cyclohexane, and dichloromethane.
 12. The method according to claim 11,wherein the reducing agent is a thiol reducing agent.
 13. The methodaccording to claim 1, wherein the selenium-containing compound is aselenium-containing compound of one of the following chemical formulas:


14. The method according to claim 2, wherein the selenium-containingcompound is a selenium-containing compound of one of the followingchemical formulas:


15. The method according to claim 3, wherein the selenium-containingcompound is a selenium-containing compound of one of the followingchemical formulas:


16. The method according to claim 4, wherein the selenium-containingcompound is a selenium-containing compound of one of the followingchemical formulas:


17. The method according to claim 11, wherein the selenium-containingcompound is a selenium-containing compound of one of the followingchemical formulas: